1. B Cell Activation and Antibody Production Lecture 15

2. Overview of B Cell Development, Activation, Antibody Production

3. B Cell Antigens

4. B Cell Responses to Thymus- Dependent Antigens (T Cell- Dependent Antibody Responses)

5. Primary and Secondary Antibody Responses

6. Phases of the Humoral Immune Response

7. T Cell Epitope B Cell Epitope Antigen A T-Dependent Antigen Must Contain Both B and T Cell Epitopes LINKED RECOGNITION

8. (B Cells) (T Cells) Follicles

9. Activation of B Cells by Antigen and Complement 1. Biochemical Signals 2. Endocytosis of Antigen

10. Antigen Recognition Phase of T-Dependent Antibody Response

11. Interactions of B Cells with Helper T Cells Initial ContactT-B Conjugate TEM Picture Note the broad area of membrane contact between B and T Cells. B Cell T Cell

12. Helper T Cell-Dependent Activation Of B Lymphocytes

13. B-Cell Activation by Thymus-Dependent Antigens Cytokines Linked Recognition C’R

14. Activated B Cells (Following Interaction with T H Cells Extra-follicular SiteFollicle Antibody Secreting Cells Germinal Center Antibodies

15. Late Events in T Cell-Dependent Antibody Responses-Germinal Center Reaction Affinity Maturation –Somatic Hypermutation Generation of Memory B Cells Affinity Maturation –Somatic Hypermutation Generation of Memory B Cells

16. Somatic Hypermutation and Affinity Maturation of Antibodies Affinity maturation is the process that leads to increased affinity of antibodies for a particular antigen as a result of somatic mutation in the Ig genes followed by selective survival of B cells producing the antibodies with the highest affinity

17. Affinity Maturation in Antibody Responses

18. Selection of High Affinity B Cells in Germinal Center

19. Phases of the Humoral Immune Response to T-Dependent Antigen

20. Anatomy of Humoral Immune Responses

22. Antibody Isotype Switching

23. Isotype Switching Under the Influence of Helper T Cell-Derived Cytokines

24. Mechanism of Ig Isotype Switching

25. CD4 T Cell-Dependent Effects in Antibody Responses Memory B Cell Development Isotype Switching Affinity Maturation Memory B Cell Development Isotype Switching Affinity Maturation

27. Thymus- Independent Antigens

28. B-Cell Activation by Thymus-Independent and Dependent Antigens Most TI antigens are polyvalent and induce maximal Crosslinking of membrane Ig on B cells, without a Need for T cell help.

29. Features of Antibody Responses to T-Dependent and T-Independent Antigens

30. Antibody Response to T-Dependent Antigens Role of Helper T Cells –Cytokines –CD40/CD40L interactions Isotype Switching –Switch Recombination –Cytokines and Isotypes Affinity Maturation –Somatic hypermutation –Selection for B cells which produce High Affinity Antibodies Memory B Cells Role of Helper T Cells –Cytokines –CD40/CD40L interactions Isotype Switching –Switch Recombination –Cytokines and Isotypes Affinity Maturation –Somatic hypermutation –Selection for B cells which produce High Affinity Antibodies Memory B Cells

31. Antibody Effector Functions

32. Effector Functions of Antibodies

33. Neutralization of Microbes by Antibodies

34. Neutralization of Toxins by Antibodies

35. Opsonization of Microbes by Antibodies

36. Antibody-Dependent Cellular Cytotoxicity (ADCC)

37. Functions of Complement

38. Complement-Mediated Lysis of E. coli Alive Killed

40. Cellular Interactions in Immune Responses

41. The Immune Response: A Summary

45. WHY can immune system recognize so many different epitopes?? Antibody heavy and light chains are composed of gene segments Variable regions are unique A limited variety of constant region sequences are used They must be rearranged into functional genes before they can be transcribed

46. p. 106

47. Organization of Ig genes Germline DNA- gene segments surrounded by noncoding regions These are rearranged to form functional genes Light chains- V, J and C segments Heavy chain- V, D, J, C V regions rearrange first A single V can rearrange to more than one C

48. Multigene families  or In humans: 40 V , 5 J , 1 C  Similar number of genes in humans; this is rare in mice Heavy-chain gene families are similar but more complex (D segment) CH regions formed from exons

49. One of many possible combinations p. 111

50. Heavy chain DNA D-J and V-DJ rearrangements must occur separately On a mature B cell, both mIgM and mIgD are expressed on the cell surface

51. How does rearrangement occur? Each V, D and J is flanked by RSS (Recombination signal sequences) Mechanism is controlled by RAG-1 and RAG-2 proteins and an enzyme TdT If any of these proteins is defective no mature B cells can form; nor T cells

52. p. 112

53. “Junctional flexibility” contributes to diversity But not all rearrangements are “productive” p. 115

54. B cells are diploid and contain chromosomes from both parents However, heavy chain genes are rearranged from only one chromosome, as are light chain genes. Therefore, any one B cell will contain one V H and one V L (antigen specificity) How? Allelic exclusion (Yancopoulos and Alt, 1986)

55. Model for allelic exclusion p. 116

56. Generation of antibody diversity (why are there so many possible antigen combining sites?)

57. Multiple germline gene segments In human germline: 51 V H, 27 D, 6 J H 40 V , 5 J  30 V, 4 J

58. Combinatorial V-J and V-D-J joining 57 V X 27 D X 6 J= 8262 possible combinations for VDJ joining 40 V X 5J = 200 possible V  120 possible V 8262 X (200+120) = 2.64 X 10 6 possible combinations Without taking into account other sources of diversity

59. Junctional flexibility in V-J or V-D-J junction Additional nucleotides added at junctions (P or N addition), if a single-stranded region is created during the joining process Somatic hypermutation mutations occur AFTER rearrangement tends to occur in CDR regions affects antigen affinity (tends to increase): “affinity maturation” occurs in B but not T cells

60. Class switching After antigen stimulation heavy-chain DNA can rearrange so VDJ joins to any isotype Cytokines help determine the isotype IgG2a or IgG3 (mice): IFN-  IgM: IL-2, IL-4, IL-5 IgE: IL-4

61. p. 122

62. Membrane-bound or secreted? Alternative splicing, p. 124

63. Mature B cells express both mIgM and mIgD No switch site between C  and C  The VDJC  C  contains 4 polyadenylation sites mIgM or mIgD can be generated depending on which polyadenylation site is used

64. Regulatory elements of transcription Promoters Enhancers Gene silencers Gene rearrangement brings enhancers close to the promoter they influence

65. Why aren’t Igs produced in B cells? In T cells a protein may bind to the  -enhancer and prevent V-J joining Arrangement of immunoglobulin genes (and formation from exons) and greatly facilitated formation of genetically engineered antibodies